Improved Immune Monitoring and Correlates of Therapy Durability

Establish blood-based T cell biomarkers using IsoCode polyfunctional profiling to predict therapy durability and patient survival.

IsoCode Data

FOCiS 2018 Abstract: Single-Cell Polyfunctionality of CD4+ T Cells Shows Promise as a Predictor of Overall Survival of Pancreatic Cancer Patients Treated with GVAX Vaccine

The therapeutic GVAX vaccine boosts the body’s immune system T-cells to fight pancreatic cancer. To identify clinical correlates of polyfunctional response of T-cells that produce 2+ cytokines per single cell to GVAX vaccine, we employed 32-plex single-cell cytokine profiling platform to evaluate T-cell polyfunctionality in patients with pancreatic cancer who had GVAX vaccination.

CD4+ T-cells were isolated with anti-CD4 microbeads from pre- and post-vaccination PBMCs and stimulated with anti-CD3/CD28 at 37°C, 5% CO2 for 24 hours. After stimulation, cells were loaded into a single-cell IsoCode chip containing ~12000 microchambers, each pre-patterned with a complete copy of a 32-plex antibody array. Protein secretions from ~1000 individual CD4+ T-cells were analyzed after 16-hour-on-chip incubation. Polyfunctional profile was assessed by the 5 functional groups: effector (Granzyme B, IFN-γ, MIP-1α, Perforin, TNF-α, TNF-β), stimulatory (GM-CSF, IL-2, IL-5, IL-7, IL-8, IL-9, IL-12, IL-15, IL-21), regulatory (IL-4, IL-10, IL-13, IL-22, TGF-β1, sCD40L, sCD137), inflammatory (IL-1b, IL-6, IL-17A, IL-17F, MCP-1, MCP-4), and chemoattractive (CCL-11, IP-10, MIP-1β, RANTES).

The single-cell analysis demonstrates a marked upregulation of polyfunctional CD4+ T-cells across 5 patients after GVAX vaccination compared to pre-vaccinated CD4+ T-cells. The enhanced polyfunctional strength index (PSI) of CD4+ T-cells by GVAX was predominated by antitumor-associated effector proteins including Granzyme B, IFN-Υ, MIP-1α, Perforin and TNF-α, mixed with small amounts of MIP-1β, sCD137 secretions. Most importantly, post- versus pre-vaccination fold-change of PSI was significantly associated with patient overall survival (P = 0.001), indicating a potential of PSI in predicting GVAX vaccine efficacy and outcomes of patients with pancreatic cancer.

Publications

Lu Y, Xue Q, Eisele MR, Sulistijo E, Brower K, Han L, Amir ED, Pe’er D, Miller-Jensen K, and Fan R. Highly Multiplexed Profiling of Immune Effector Functions Reveals Deep Functional Heterogeneity in Response to Pathogenic Ligands. Proc. Natl. Acad. Sci., 112 (7), 607-615 (2015).

Lu Y, Chen JJ, Mu L, Xue Q, Wu Y, Wu PH, Li J, Vortmeyer AO, Miller-Jensen K, Wirtz D, and Fan R. High-throughput Secretomic Analysis of Single Cells to Assess Functional Cellular Heterogeneity. Analytical Chemistry, 85 (4), 2548– 2556 (2013).

Ma C, Fan R, Ahmad H, Shi Q, Comin-Anduix B, Chodon T, Koya RC, Liu CC, Kwong GA, Radu CG, Ribas A, and Heath JR. A Clinical Microchip for Evaluation of Single Immune Cells Reveals High Functional Heterogeneity in Phenotypically Similar T Cells. Nature Medicine, 17, 738-743 (2011).

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